Phenethyl-thiourea compounds and use

ABSTRACT

Novel phenylethyl-thiourea (PHET) compounds as inhibitors of reverse transcriptase and effective agents for the treatment of HIV infection, including mutant, drug-sensitive, drug-resistant, and multi-drug resistant strains of HIV.

This is a division of application Ser. No. 09/338,090, filed Jun. 23,1999 now U.S. Pat. No. 6,207,688.

FIELD OF THE INVENTION

The invention relates to inhibitors of reverse transcriptase effectiveagainst HIV, including mutant strains of HIV, and effective in thetreatment of multi-drug resistant HIV infection.

BACKGROUND OF THE INVENTION

Agents currently used to treat HIV infection attempt to blockreplication of the HIV virus by blocking HIV reverse transcriptase or byblocking HIV protease. Three categories of anti-retroviral agents inclinical use are nucleoside analogs (such as AZT), protease inhibitors(such as nelfinavir), and the recently introduced non-nucleoside reversetranscriptase inhibitors (NNI), such as nevirapine.

The recent development of potent combination anti-retroviral regimenshas significantly improved prognosis for persons with HIV and AIDS.Combination therapies may be a significant factor in the dramaticdecrease in deaths from AIDS (decrease in death rate as well as absolutenumber). The most commonly used combinations include two nucleosideanalogs with or without a protease inhibitor.

Nevirapine is currently the only NNI compound which has been used incombination with AZT and/or protease inhibitors for the treatment ofHIV. A new series of effective drug cocktails will most likely involveother NNIs in combination with nucleoside and protease inhibitors as atriple action treatment to combat the growing problem of drug resistanceencountered in single drug treatment strategies.

The high replication rate of the virus unfortunately leads to geneticvariants (mutants), especially when selective pressure is introduced inthe form of drug treatment. These mutants are resistant to theanti-viral agents previously administered to the patient. Switchingagents or using combination therapies may decrease or delay resistance,but because viral replication is not completely suppressed in singledrug treatment or even with a two drug combination, drug-resistant viralstrains ultimately emerge. Triple drug combinations employing one (ortwo) nucleoside analogs and two (or one) NNI targeting RT provide a verypromising therapy to overcome the drug resistance problem. RT mutantstrains resistant to such a triple action drug combination would mostlikely not be able to function.

Dozens of mutant strains have been characterized as resistant to NNIcompounds, including L1001, K103N, V106A, E138K, Y181C and Y188H. Inparticular, the Y181C and K103N mutants may be the most difficult totreat, because they are resistant to most of the NNI compounds that havebeen examined.

Recently, a proposed strategy using a knock-out concentration of NNIdemonstrated very promising results. The key idea in this strategy is toadminister a high concentration of NNI in the very beginning stages oftreatment to reduce the virus to undetectable levels in order to preventthe emergence of drug-resistant strains. The ideal NNI compound foroptimal use in this strategy and in a triple action combination mustmeet three criteria:

1) very low cytotoxicity so it can be applied in high doses;

2) very high potency so it can completely shut down viral replicationmachinery before the virus has time to develop resistant mutant strains;and

3) robust anti-viral activity against current clinically observed drugresistant mutant strains.

Novel NNI designs able to reduce RT inhibition to subnanomolarconcentrations with improved robustness against the most commonlyobserved mutants and preferably able to inhibit the most troublesomemutants are urgently needed. New antiviral drugs will ideally have thefollowing desired characteristics: (1) potent inhibition of RT; (2)minimum cytotoxicity; and (3) improved ability to inhibit known, drugresistant strains of HIV. Currently, few anti-HIV agents possess all ofthese desired properties.

Two non-nucleoside inhibitors (NNI) of HIV RT that have been approved bythe U.S. Food and Drug Administration for licensing and sale in theUnited States are nevirapine (dipyridodiazepinone derivative) anddelavirdine (bis(heteroaryl)piperazine (BHAP) derivative. BHAP U-90152).Other promising new non-nucleoside inhibitors (NNIs) that have beendeveloped to inhibit HIV RT include dihydroalkoxybenzyloxopyrimidine(DABO) derivatives, 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine(HEPT) derivatives, PHETrahydrobenzondiazepine (TIBO),2′,5′-Bis-O(tert-butyldimethylsilyl)-3′-spiro-5″-(4″-amino-1″,2″-oxathiole-2″,2′-dioxide)pyrimidine (TSAO), oxathiin carboxanilidederivatives, quinoxaline derivatives, thiadiazole derivatives, andphenethylthiazolylthiourea (PETT) derivatives.

NNIs have been found to bind to a specific allosteric site of HIV-RTnear the polymerase site and interfere with reverse transcription byaltering either the conformation or mobility of RT, thereby leading to anoncompetitive inhibition of the enzyme (Kohlstaedt, L. A. et al.,Science, 1992, 256, 1783-1790).

A number of crystal structures of RT complexed with NNIs have beenreported (including α-APA, TIBO, Nevirapine, and HEPT derivatives), andsuch structural information provides the basis for furtherderivatization of NNI aimed at maximizing binding affinity to RT.However, the number of available crystal structures of RT NNI complexesis limited.

Given the lack of structural information, alternate design proceduresmust be relied upon for preparing active inhibitors such as PETT andDABO derivatives. One of the first reported strategies for systematicsynthesis of PETT derivatives was the analysis of structure-activityrelationships independent of the structural properties of RT and led tothe development of some PETT derivatives with significant anti-HIVactivity (Bell, F. W. et al., J Med. Chem., 1995, 38, 4929-4936;Cantrell, A. S. et al., J. Med. Chem., 1996, 39, 4261-4274).

A series of selected phenethylthiazolylthiourea (PETT) derivativestargeting the NNI binding site of HIV reverse transcriptase (RT) weresynthesized and tested for anti-human immunodeficiency virus (HIV)activity. The structure based design and synthesis of these PETTderivatives were aided by biological assays and their anti-HIV activity.Some of these novel derivatives were more active than AZT or Troviridineand abrogated HIV replication at nanomolar concentrations without anyevidence of cytotoxicity. These compounds are useful in the treatment ofHIV infection, and have particular efficacy against mutant strains,making them useful in the treatment of multi-drug resistant, HIV.

SUMMARY OF THE INVENTION

The invention provides phenethyl-thiourea (PHET) compounds asnon-nucleoside inhibitors (NNI) of HIV reverse transcriptase, andparticularly methyl-phenethyl-thiourea compounds as particularly potentinhibitors. The PHET compounds, compositions, and methods of theinvention are useful in the treatment of HIV infection, with particularefficacy against multiple strains of HIV, including multi-drug resistantmutant strains.

The PHET compounds, compositions, and methods of the invention areuseful for inhibiting reverse transcriptase activity and inhibitingreplication of multiple strains of HIV, including therapy-naive,drug-resistant, and multi-drug resistant mutant strains. In particular,the PHET compounds of the invention are useful for treating retroviralinfection in a subject, such as an HIV-1 infection, by administration ofthe PHET compounds of the invention, for example, in a pharmaceuticalcomposition.

The PHET compounds of the invention contain a phenyl ring as shown inFormula I. The phenyl ring is preferably substituted (R_(n)) with anelectron-donating group at the para position, such as methyl or methoxy.An exemplary, and particulary preferred PHET pound of the invention isHI-244, having the structure shown in Formula II.

The PHET compounds and compositions useful in the invention exhibit:

1. very low cytotoxicity;

2. very high potency; and

3. potent activity against at least one clinically observed drugresistant mutant strain.

Specific compounds and methods of the invention are described more fullyin the Detailed Description and in the Examples below.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a composite binding pocket of the NNI binding site of HIV-1RT illustrated as grid lines representing the collective van der Waalssurface; a stick model of the docked compound HI-244 is shown; and

FIG. 1B is a Connolly surface representation of HI-244 in the NNIbinding site; residues in contact with the compound are labeled andshown in the stick model.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

When used herein, the following terms have the indicated meanings:

“NNI” means non-nucleoside inhibitor. In the context of the invention,non-nucleoside inhibitors of HIV reverse transcriptase (RT) are defined.

“Mutant HIV” means a strain of HIV having one or more mutated or alteredamino acids as compared with wild type.

“Multi-Drug Resistant HIV” means one or more HIV strain which isresistant to treatment with one or more chemotherapeutic agent.

“Therapeutically effective amount” is a dose which provides sometherapeutic benefit on administration, including, in the context of theinvention, reduced viral activity or viral load in a patient, and alsoincluding inhibition of viral RT activity and/or replication of virus.

Compounds of the Present Invention

Compounds of the present invention are phenethyl-thiourea (PHET)compounds useful as non-nucleoside inhibitors of RT having the formulaI:

The phenyl ring may be substituted or unsubstituted (R_(n)), and ispreferably substituted with an electron donating group, most preferablyat the para position. For example, R can be H, methyl, methoxy, halo,and the like, and most preferably is methyl. The value of n can be 0 to6, and preferably is 1.

R₁ is a cyclic moiety, which may be substituted or not (X), such asphenyl, pyridyl, piperidinyl, piperonyl, morphorlyl, furyl, and thelike, and can be, for example, cyclo(C₃-C₁₂) alkyl, cyclo(C₃-C₁₂)alkenyl, isothiazolyl, tetrazolyl, triazolyl, pyridyl, imidazolyl,phenyl, napthyl, benzoxazolyl, benzimidazolyl, thiazolyl, oxazolyl,benzothiazolyl, pyrazinyl, pyridazinyl, thiadiazolyl, benzotriazolyl,pyrolyl, indolyl, benzothienyl, thienyl, benzofuryl, quinolyl,isoquinolyl, pyrazolyl, and the like.

In one preferred embodiment, R₁ is pyridyl, optionally substituted (X)with one or more substituents, for example, with an alkyl, alkoxy, halo,or hydroxy group. More preferably, R₁ is pyridyl substituted with ahalogen such as bromine or chlorine. An exemplary compound of theinvention is N-[2-(4-methylphenethyl)]-N′-[2-(5-bromopyridyl)]-thiourea(HI-244).

The compounds of the invention preferably bind to a specific allostericsite of HIV-RT near the polymerase site and interfere with reversetranscription, for example, by altering either the conformation ormobility of RT.

Acid Salts

The compounds of the invention may also be in the form ofpharmaceutically acceptable acid addition salts. Pharmaceuticallyacceptable acid addition salts are formed with organic and inorganicacids.

Examples of suitable acids for salt formation are hydrochloric,sulfuric, phosphoric, acetic, citric, oxalic, malonic, salicylic, malic,gluconic, fumaric, succinic, asorbic, maleic, methanesulfonic, and thelike. The salts are prepared by contacting the free base form with asufficient amount of the desired acid to produce either a mono or di,etc. salt in the conventional manner. The free base forms may beregenerated by treating the salt form with a base. For example, dilutesolutions of aqueous base may be utilized. Dilute aqueous sodiumhydroxide, potassium carbonate, ammonia, and sodium bicarbonatesolutions are suitable for this purpose. The free base forms differ fromtheir respective salt forms somewhat in certain physical properties suchas solubility in polar solvents, but the salts are otherwise equivalentto their respective free base forms for purposes of the invention. Useof excess base where R is hydrogen gives the corresponding basic salt.

Methods of Using the Compounds of the Invention

The compounds of the invention are useful in methods for inhibitingreverse transcriptase activity of a retrovirus. Retroviral reversetranscriptase is inhibited by contacting RT in vitro or in vivo, with aneffective inhibitory amount of a compound of the invention. Thecompounds of the invention also inhibit replication of retrovirus,particularly of HIV, such as HIV-1. Viral replication is inhibited, forexample, by contacting the virus with an effective inhibitory amount ofa compound of the invention.

The methods of the invention are useful for inhibiting reversetranscriptase and/or replication of multiple strains of HIV, includingmutant strains, and include treating a retroviral infection in asubject, such as an HIV-1 infection, by administering an effectiveinhibitory amount of a compound or a pharmaceutically acceptable acidaddition salt of a compound of the Formula I. The compound or inhibitorof Formula I is preferably administered in combination with apharmaceutically acceptable carrier, and may be combined with specificdelivery agents, including targeting antibodies and/or cytokines. Thecompound or inhibitor of the invention may be administered incombination with other antiviral agents, immunomodulators, antibioticsor vaccines.

The compounds of Formula I can be administered orally, parentally(including subcutaneous injection, intravenous, intramuscular,intrasternal or infusion techniques), by inhalation spray, topically, byabsorption through a mucous membrane, or rectally, in dosage unitformulations containing conventional non-toxic pharmaceuticallyacceptable carriers, adjuvants or vehicles. Pharmaceutical compositionsof the invention can be in the form of suspensions or tablets suitablefor oral administration, nasal sprays, creams, sterile injectablepreparations, such as sterile injectable aqueous or oleagenoussuspensions or suppositories. In one embodiment, the PHET compounds ofthe invention can be applied intravaginally and/or topically, forexample in gel form, for prevention of heterosexual transmission of HIV.

For oral administration as a suspension, the compositions can beprepared according to techniques well-known in the art of pharmaceuticalformulation. The compositions can contain microcrystalline cellulose forimparting bulk, alginic acid or sodium alginate as a suspending agent,methylcellulose as a viscosity enhancer, and sweeteners or flavoringagents. As immediate release tablets, the compositions can containmicrocrystalline cellulose, starch, magnesium stearate and lactose orother excipients, binders, extenders, disintegrants, diluents andlubricants known in the art.

For administration by inhalation or aerosol, the compositions can beprepared according to techniques well-known in the art of pharmaceuticalformulation. The compositions can be prepared as solutions in saline,using benzyl alcohol or other suitable preservatives, absorptionpromoters to enhance bioavailability, fluorocarbons or othersolubilizing or dispersing agents known in the art.

For administration as injectable solutions or suspensions, thecompositions can be formulated according to techniques well-known in theart, using suitable dispersing or wetting and suspending agents, such assterile oils, including synthetic mono- or diglycerides, and fattyacids, including oleic acid.

For rectal administration as suppositories, the compositions can beprepared by mixing with a suitable non-irritating excipient, such ascocoa butter, synthetic glyceride esters or polyethylene glycols, whichare solid at ambient temperatures, but liquefy or dissolve in the rectalcavity to release the drug.

Dosage levels of approximately 0.02 to approximately 10.0 grams of acompound of the invention per day are useful in the treatment orprevention of retroviral infection, such as HIV infection, AIDS orAIDS-related complex (ARC), with oral doses 2 to 5 times higher. Forexample, HIV infection can be treated by administration of from about0.1 to about 100 milligrams of compound per kilogram of body weight fromone to four times per day. In one embodiment, dosages of about 100 toabout 400 milligrams of compound are administered orally every six hoursto a subject. The specific dosage level and frequency for any particularsubject will be varied and will depend upon a variety of factors,including the activity of the specific compound the metabolic stabilityand length of action of that compound, the age, body weight, generalhealth, sex, and diet of the subject, mode of administration, rate ofexcretion, drug combination, and severity of the particular condition.

The compound of Formula I can be administered in combination with otheragents useful in the treatment of HIV infection, AIDS or ARC. Forexample, the compound of the invention can be administered incombination with effective amounts of an antiviral, immunomodulator,anti-infective, or vaccine. The compound of the invention can beadministered prior to, during, or after a period of actual or potentialexposure to retrovirus, such as HIV.

Methods of Making the Compounds of the Invention

The compounds of the invention may be prepared as shown in Schemes 1 and2. In general, an appropriate phenethylamine or pyridylethylamine(R₁—NH₂) is reacted with 1,1′-thiocarbonyl-diimidazole in acetonitrilesolvent at ambient temperature for approximately 12 hours to form athiocarbonyl reagent. The reaction product is then condensed with asubstituted or non-substituted phenylamine in an aprotic solvent such asdimethyl-formamide (DMF) at elevated temperature, such a 100° C., for anextended period of time such as about 15 hours. The desired PHETcompound is purified by column chromatography.

The PHET compounds of the invention can be synthesized as describedabove, or by other, known synthetic methods.

EXAMPLES

The invention may be further clarified by reference to the followingExamples, which serve to exemplify the embodiments, and not to limit theinvention in any way.

Example 1 Synthesis and Characterization of Thiourea Inhibitors

In the present study, we replaced the pyridyl ring of trovirdine with asubstituted or unsubstituted phenyl group that fits well with the Wing 2region of the NNI binding pocket. The PHET compounds were synthesized asshown in Scheme 1, in which a thiocarbonyl reagent was prepared fromphenethylamine or pyridylethylamine and 1,1′-thiocarbonyl-diimidazole inacetonitrile solvent at room temperature for 12 hours, and condensedwith the appropriate 2-phenyl amino compounds in dimethyl formamide(DMF) at 100° C. for 15 hours. After work up, the derivatives werepurified by column chromatography. Trovirdine, a comparative standard,was synthesized according to the method described in Bell et.al., 1995,J Med. Chem 38:4929.

Characterization of Synthesized Compounds:

Proton and carbon nuclear magnetic resonance spectra were recorded on aVarian spectrometer using an automatic broad band probe. Unlessotherwise noted, all NMR spectra were recorded in CDCl₃ at roomtemperature. The chemical shifts reported are in parts per millionrelative to tetramethyl silane as standard. The multiplicity of thesignals were designated as follows: s, d, dd, t, q, m which correspondsto singlet, doublet, doublet of doublet, triplet, quartet and multipletrespectively. UV spectra were recorded from a Beckmann Model #DU 7400UV/V is spectrometer using a cell path length of 1 cm. Fourier TransformInfra Red spectra were recorded using an FT-Nicolet model Protege #460instrument. The infra red spectra of the liquid samples were run as neatliquids using KBr discs. Mass spectrum analysis was conducted usingeither a Finnigan MAT 95 instrument or a Hewlett-Packard Matrix AssistedLaser Desorption (MALDI) spectrometer model #G2025A The matrix used inthe latter case was cyano hydoxy cinnamic acid. Melting points weredetermined using a Melt John's apparatus and uncorrected. Elementalanalysis were was performed by Atlantic Microlabs (Norcross, Ga). Columnchromatography was performed using silica gel obtained from the BakerCompany. The solvents used for elution varied depending on the compoundand included one of the following: ethyl acetate, methanol, chloroform,hexane, methylene chloride and ether. Characterizataion data for thesynthesized compounds is shown below:

N-[2-(4-methoxyphenethyl)]-N′-[2-(5-bromopyridyl)]thiourea (HI-238)

yield: 85%, mp.178-179° C.; UV:(MeOH) λmax: 205, 226, 275, 305 nm;IR(KBr) ν 3221, 3159, 3042, 2931, 2827, 1587, 1510, 1464, 1311, 1225,1165, 1088, 1034, 820, 773, 708 cm⁻¹; ¹HNMR (CDCl₃) δ 11.30 (bs,1H),7.87 (bs, 1H), 8.00-7.99 (d, 1H), 7.21-7.18 (dd, 1H), 6.95-6.92 (d,2H),6.88-6.85(d, 2H), 4.00-3.93 (q, 2H), 3.81 (s, 3H), 2.96-292(t, 2H);¹³C(CDCl₃) δ 178.1, 158.0, 151.9, 145.8, 140.7, 130.6, 128.6, 113.8,113.7, 112.1, 55.1, 46.9 and 33.8; Maldi Tof mass: 366 (M+1), Calculatedmass: 365; Anal. (C₁₅H₁₆BrN₃OS) C, H, N, S;

N-[2-(4-fluorophenethyl)]-N′-[2-(5-bromopyridyl)]thiourea (HI-242)

yield: 69%; mp 177-178° C.; UV:(MeOH) λmax: 208, 211, 274, 306 nm,IR(KBr) ν 3456, 3213, 3155, 3086, 3028, 2868, 1595, 1560, 1533, 1477,1336, 1308, 1238, 1211, 1173, 1136, 1092, 1026, 933, 869, 827, 791,7741, 694 cm⁻¹;¹HNMR (CDCl₃) δ 11.29 (bs, 1H), 9.27 (bs, 1H), 8.04-8.03(dd, 1H), 7.73-7.69(dd, 1H), 7.271-7.22(m, 1H), 7.04-6.99 (m, 3H),6.83-6.79 (d, 1H), 4.03-3.96 (q, 2H), 3.02-2.97 (t, 2H); ¹³C(CDCl₃) δ179.1, 163.2, 151.6, 146.3, 141.2, 134.3, 130.3, 130.2, 115.2, 113.5,112.8, 47.0 and 34.1; ¹⁹F(CDCl₃) δ-40.55 (m), Maldi Tof mass: 354.8(M+1), Calculated mass: 354; Anal. (C₁₄H₁₃BrFN₃S) C, H, N, S;

N-[2-(4-bromophenethyl)]-N′-[2-(5-bromopyridyl)]thiourea (HI-243)

yield: 75%; mp 184-185° C.; UV (MeOH) λmax: 207, 257, 276, 306 nm,IR(KBr) ν 3454, 3221, 3153, 3086, 3022, 2929, 1595, 1560, 1531,1471,1402, 1338, 1304, 1227, 1169, 1091, 1072, 1013, 820, 708 cm⁻¹;¹HNMR (CDCl₃) δ 11.28 (bs, 1H), 9.21 (bs, 1H), 8.04-8.03 (dd. 1H),7.46-7.43(dd, 2H), 7.18-7.14 (m, 2H), 6.81-6.78 (d, 2H), 4.03-3.96 (q,2H), 3.00-2.96 (t, 2H), ¹³C(CDCl₃) δ 179.2, 151.6, 146.3, 141.3, 133.6,131.6, 130.6, 113.5, 112.9, 46.7 and 34.3; Maldi Tof mass: 416 (M+1),Calculated mass: 415; Anal. (C₁₄H₁₃Br₂N₃S) C, H, N, S, Br;

N-[2-(4-methylphenethyl)]-N′-[2-(5-bromopyridyl)]thiourea (HI-244)

yield: 62%; mp. 159-162° C.; UV (MeOH) λmax 212, 275, 305 nm; IR (KBr) ν3439, 3228, 3161, 3086, 2917, 2866, 1595, 1543, 1466, 1307, 1265, 1229,1188, 1140 , 1003, 829, 812, 777 cm⁻¹; ¹HNMR (CDCl₃) δ 11.27 (bs, 1H),9.07 (bs, 1H), 8.01-8.00(s, 1H), 7.71-7.67 (d, 1H), 7.19-7.12 (m, 4H),6.78-6.75 (d, 1H), 4.03-3.97 (q, 2H), 2.99-2.95 (t, 2H), 2.36 (s, 3H);¹³C(CDCl₃) δ 178.9, 151.6, 146.4, 141.1, 136.1, 135.5, 129.2, 128.7,113.4, 112.7, 47.2, 34.4 and 2 1.1; Maldi Tof mass: 351 (M+1),Calculated mass: 350; Anal. (C₁₅H₁₆Br N₃S) C, H, N, S;

N-[2-(4-chlorophenethyl)]-N′-[2-(5-bromopyridyl)]thiourea (HI-255)

yield: 71%; mp. 180-183° C.; UV (MeOH) λmax206, 209, 219, 256, 275, 305nm; IR(KBr) ν 3221, 3153, 3086, 2931, 2931, 1674, 1593, 1562, 1533,1473, 1406, 1340, 1304, 1265, 1227, 1169, 1138, 1092, 1016, 820, 752,714 cm⁻¹; ¹HNMR (CDCl₃) δ 11.40 (bs, 1H), 9.34 (bs, 1H), 8.15-8.14(s,1H), 7.84-7.80 (d, 1H), 7.42-740 (m, 2H), 7.40-7.33 (m, 2H), 7.33-7.30(m, 1H), 6.92-6.89 (d, 1H), 4.10-4.07 (q, 2H), 3.13-3.08 (t, 2H);¹³C(CDCl₃) δ 179.2, 151.6, 146.3, 141.3, 137.1, 130.2, 128.6, 113.5,112.8, 46.8 and 34.2; Maldi Tof mass: 372 (M+1), Calculated mass: 371.0;Anal. (C₁₄H₁₃Br ClN₃S) C, H, N, S;

N-[2-(5-Bromopyridinyl)]-N′-[2-(Phenethyl)]thiourea (HI-275)

mp: 162-163° C.; UV(MeOH) λ_(max): 207, 276, 306 nm; IR(KBr) ν 3216,3160, 3083, 3025, 2925, 2861, 1159, 1552, 1529, 1475, 1357, 1311, 1226,1166, 1091, 1070, 1002, 864, 823, 746, 698, 561, 511 cm⁻¹; ¹HNMR (CDCl₃)δ 11.31 (bs, 1H), 9.42 (bs, 1H), 7.99-7.98 (d, 1H), 7.70-7.66 (dd, 1H),7.37-7.26 (m, 5H), 6.84-6.81 (d, 1H), 4.06-4.00 (q, 2H), 3.04-2.99 (t,2H); ¹³CNMR (CDCl₃) δ 179.0, 151.7, 146.2, 141.1, 138.6, 128.8, 128.5,126.6, 113.5, 112.7, 47.0, 34.8;

N-[2-(4-hydroxyphenethyl)]-N′-[2-(5-bromopyridyl)]thiourea (HI-256)

yield: 71%; mp 159-162° C.; UV (MeOH) λmax 205, 208, 274, 305 nm; IR(KBr) ν 3224, 3159, 3089, 3041, 2933, 2870, 1595, 1558, 1533, 1514,1332, 1306, 1265, 1227, 1186, 1136, 1094, 1007, 910, 862, 827, 708 cm⁻¹;¹HNMR (DMSO-d₆) δ 11.30 (bs, 1H), 10.17 (bs, 1H), 8.78 (bs, 1H),8.03-8.02(s, 1H), 7.67-7.65(d, 1H), 7.11-7.09 (d, 2H), 7.03-6.99 (d,1H), 6.82-6.80 (d, 2H), 3.96-3.90 (q, 2H), 2.92-2.83 (t, 2H), ¹³C(CDCl₃)δ 178.9, 155.4, 151.9, 145.4, 140.3, 129.2, 128.9, 114.9, 113.9, 111.6,46.7 and 33.5. Maldi Tof mass: 353 (M+1), Calculated mass: 352; Anal.(C₁₄H₁₄BrN₃OS) C, H, N, S, Br.

Example 3 Antiviral Activity of Substituted Thiourea Compounds

Purified RT Assays for Anti-HIV Activity

The synthesized compounds were tested for RT inhibitory activity(IC₅₀[rRT]) against purified recombinant HIV RT using the cell-freeQuan-T-RT system (Amersham, Arlington Heights, Ill.), which utilizes thescintillation proximity assay principle as described in Bosworth, etal., 1989, Nature 341:167-168. In the assay, a DNA/RNA template is boundto SPA beads via a biotin/strepavidin linkage. The primer DNA is a16-mer oligo(T) which has been annealed to a poly(A) template. Theprimer/template is bound to a strepavidin-coated SPA bead.

³H-TTP is incorporated into the primer by reverse transcription. Inbrief, ³H-TTP, at a final concentration of 0.5 μCi/sample, was dilutedin RT assay buffer (49.5 mM Tris-Cl, pH 8.0, 80 mM KCl, 10 Mm MgCl₂, 10mM DTT, 2.5 mM EGTA, 0.05% Nonidet-P-40), and added to annealed DNA/RNAbound to SPA beads. The compound being tested was added to the reactionmixture at 0.001 μM-100 μM concentrations. Addition of 10 mU ofrecombinant HIV RT and incubation at 37° C. for 1 hour resulted in theextension of the primer by incorporation of ³H-TTP. The reaction wasstopped by addition of 0.2 ml of 120 mM EDTA. The samples were countedin an open window using a Beckman LS 7600 instrument and IC₅₀ valueswere calculated by comparing the measurements to untreated samples.

In addition, the anti-HIV activity of the compounds was measured bydetermining their ability to inhibit the replication of the HIV-1 strainHTLVIIIB in peripheral blood mononuclear cells (PBMC) from healthyvolunteer donors, using the method described in Uckun et.al., 1998,Antimicrobial Agents and Chemotherapy 42:383. The HIV strain HTLVIIIBwas kindly provided by Dr. Neal T.Wetherall, VIROMED Laboratories, Inc.,and was propagated in CCRF-CEM cells.

Normal human peripheral blood mononuclear cells (PBMNC) fromHIV-negative donors were cultured 72 hours in RPMI 1640 supplementedwith 20% (v/v) heat-inactivated fetal bovine serum (FBS), 3%interleukin-2,2 mM L-glutairine, 25 mM HEPES, 2 μL, NAHCO, 50 mg/mLgentamicin, and 4 μg/mL phytohemagglutinin prior to exposure to HIV-1 orother HIV strain at a multiplicity of infection (MOI) of 0.1 during aone-hour adsorption period at 37° C. in a humidified 5% CO2 atmosphere.Subsequently, cells were cultured in 96-well microplates (100 μL/well;2×10⁶ cells/mL, triplicate wells) in the presence of various inhibitorconcentrations. Aliquots of culture supernatants were removed from thewells on the 7^(th) day after infection for p24 antigen p24 enzymeimmunoassays (EIA), as previously described in Erice et al., 1993,Antimicrob. Ag. Chemotherapy 37:385-838. The applied p24 EIA was theunmodified kinetic assay commercially available from CoulterCorporation/Immunotech, Inc. (Westbrook, Me.). Percent inhibition ofviral replication was calculated by comparing the p24 values from thetest substance-treated infected cells with p24 values from untreatedinfected cells (i.e, virus controls).

A Microculture tetrazolium Assay (MTA), using2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-PHETrazoliumhydroxide (XTT), was performed to evaluate the cytotoxicity of thecompounds, using the methods described, for example, in Uckun et.al.,1998, Antimicrobial Agents and Chemotherapy 42:383; and Mao et.al.,1998, Bioorg. Med. Chem. Lett. 8:2213.

Data are presented in Table 1 as the IC₅₀ values for inhibition of HIVp24 antigen production in PBMC (concentration at which the compoundinhibits p24 production by 50%).

TABLE 1

IC₅₀ IC₅₀ IC₅₀ IC₅₀ RT-MDR A17 rRT HTLV_(III)B (Y106A) (Y181C) CC₅₀Compound R₁ (μM) (μM) (μM) (μM) (μM) 1. Trovirdine Pyridyl 0.8 0.0070.02 0.5 >100 2. HI-275 Phenyl 1.3 N.D. N.D. N.D. N.D. 3. HI-2384-methoxyphenyl 0.9 0.015 N.D. N.D. >100 4. HI-242 4-flurophenyl 6.4N.D. N.D. N.D. N.D. 5. HI-255 4-chlorophenyl 2.5 20.8 N.D. N.D. N.D. 6.HI-243 4-bromophenyl 0.9 0.07 >0.001 1.0 >100 7. HI-2314-nitrophenyl >100 N.D. N.D. N.D. N.D. 8. HI-244 4-methylphenyl 0.10.007 >0.001 0.07 71 9. HI-256 4-hydroxyphenyl 87.7 3.067 N.D. N.D. >100

These data demonstrate the surprisingly potent antiviral activity ofmono-substituted phenethyl thiourea compounds, particularly substitutedwith an electron donating group at the para position of the phenyl ring.The ability of HI-244 to inhibit HIV-1 replication, including resistantstrains, in human PBMC was also evaluated using the methods describedabove. HI-244 effectively inhibited replication of the HIV-1 strainHTLV_(IIIB) in PBMC with an IC₅₀ value of 0.007 μM, which is equal tothe IC₅₀ value of trovirdine (Table 1).

HI-244 was 20-times more effective than trovirdine against the multidrugresistant HIV-1 strain RT-MDR with a V106A mutation (as well asadditional mutations involving the RT residues 74V, 41L, and 215Y) and7-times more potent than trovirdine against the NNI-resistant HIV-1strain A17 with a Y181C mutation. HI-244 was not cytotoxic to PBMC evenat a 100 μM concentration. Thus, the selectivity index for HI-244 was:

>10,000 against the wild-type HIV-1 strain HTLV_(IIIB),

>71,000 against the multidrug-resistant V106A mutant strain RT-MDR, and

>1,000 against the NNI-resistant Y181C mutant strain A17.

These findings establish the lead para-methyl substituted phenylthiourea compound HI-244 as an effective NNI against drug-sensitive,NNI-resistant, and multidrug-resistant strains of HIV-1.

As described in our prior publications, the para-substituted group lieswithin a hydrophobic region indicated by the composite NNI bindingpocket (FIG. 1A). See for example. Vig et al., 1998, Bioorg. Med. Chem,6:1789; and Sudbeck et al., 1998, Antimicro. Agents Chemother.,1998,_(—):3225-33. This region contains the aromatic rings of residuesW229 and Y188 which would interact favorably with a hydrophobic group(FIG. 1B). Therefore, the observed inhibition level of thepara-substituted compounds against the wild-type HIV-1 is generallyproportional to the hydrophobicity of the para-substituted group (Table2). The potency of the para-substitued compounds is consistent with thefollowing trend: hydrophobic group>polar group>hydrophilic group.

The para-methyl group on the HI-244 compound is predicted to residecloser to the Y181 residue (C-to Cα distance+5.5 Å) than to the V106residue (C-to-Cα distance+8.1 Å). Therefore, the Y181 mutation wouldhave reduced the potency of a para-substituted compound more than theV106 mutation would. This reasoning is consistent with the observationthat the Y181C mutant strain (A17 strain) has 10-fold resistance toHI-244 whereas the V106A mutant strain (RT-MDR) shows no resistance, andin fact shows improved activity.

TABLE 2 K_(i) ^(c) IC₅₀ ID₅₀ MS BS LIPO (calc.) rRT* p24 Compounds R₁(Å)^(a) (%)^(b) score (μM) (μM) (μM) 1. Trovirdine Pyridyl 276 84 6790.7 0.8 0.007 2. HI-275 Phenyl 274 84 672 0.8 1.3 ND 3. HI-2384-methoxyphenyl 302 84 729 1.2 0.9 0.015 4. HI-242 4-flurophenyl 284 81674 7.8 6.4 ND 5. HI-255 4-chlorophenyl 293 81 696 4.7 2.5 ND 6. HI-2434-bromophenyl 295 82 708 6.3 0.9 0.07  7. HI-231 4-nitrophenyl 301 79695 84 >100 ND 8. HI-244 4-methylphenyl 294 84 724 0.25 0.1 0.007 9.HI-256 4-hydroxyphenyl 286 82 686 104 87.7 ND *rRT is recombinant HIVreverse transcriptase ^(a)MS is the molecular surface area calculated byCommonny″s MS program ^(b)BS is burried surface, or % of the molecularsurface in contact with protein calculated by Ludi based on dockedpositions. ^(c)Ludi K_(i) values were calculated based on modifiedempirical score function in Ludi program. ND is not determined, forcompounds with IC₅₀[rRT] greater than 1.0

The apparent van der Waals contact between the compound and tyrosineresidue 181 after its mutation to a smaller cystein residue may helpexplain the observed resistance. However, we are not certain about thereason for the demonstrated superior activity of HI-244 against theV106A RT mutant relative to the wild-type RT. The V106A mutation mayenable the compound to reposition itself in the binding site, leading toimproved contacts between the para-methyl group and Y188 resulting in abetter binding affinity. In contrast, trovirdine would not benefit fromsuch a mutation due to the absence of a para-methyl group (trovirdine is20 times less potent than HI-244 against the RT-MDR strain).

With respect to trovirdine, the addition of a paramehtyl group in HI-244increases the molecular volume in the Wing 2 region of the binding siteby 18 Å³. In the NNI-resistant A17 strain (Y181C mutation), the Wing 2region of the mutant becomes larger when Y181 is mutated to a smallercystein residue, as observed in the crystal structure of the Y181Cmutant. These results showing a 7-fold higher potency of HI-244 relativeto trovirdine are consistent Faith our previously reported hypothesisthat an NNI compound containing larger (and compatible) functionalgroups at the Wing 2 region of the binding site can provide betterinhibitor activity against these HIV RT mutants.

All publications, patents, and patent documents described herein areincorporated by reference as if fully set forth. The invention describedherein may be modified to include alternative embodiments. All suchobvious alternatives are within the spirit and scope of the invention,as claimed below.

We claim:
 1. The compound of the formula


2. The compound of the formula:


3. The compound of the formula :